This invention relates to medical science particularly the treatment of sickle cell disease or thalassemia with protein C.
Protein C is a vitamin K dependent serine protease and naturally occurring anticoagulant that plays a role in the regulation of hemostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human protein C circulates as a 2-chain zymogen, but functions at the endothelial and platelet surface following conversion to activated protein C (aPC) by limited proteolysis with thrombin in complex with the cell surface membrane protein, thrombomodulin.
In conjunction with other proteins, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. In addition to its anti-coagulation functions, aPC hasanti-inflammatory effects through its inhibition of cytokine generation (e.g. TNF and IL-1) and also exerts profibrinolytic properties that facilitate clot lysis. Thus, the protein C enzyme system represents a major physiological mechanism of anti-coagulation, anti-inflammation, and fibrinolysis.
Sickle cell diseases (SCD) and thalassemia are inherited hemoglobulinopathies characterized by a structural hemoglobin defect. SCD include diseases which cause sickling of the red blood cells, and includes sickle cell anemia (which results from rwo hemoglobulin S genes), sickle xcex1-thalassemia (one hemoglobin S and one xcex1-thalassemia gene), and hemoglobin SC disease (one hemoglobin S and one hemoglobin C), and the rarer disease, hemoglobin C Harlem. Thalassemia includes xcex2-thalassemia and xcex1-thalassemia. These hereditary diseases have significant morbidity and mortality and affect individuals of African American heritage, as well as those of Mediterranean, Middle Eastern, and South East Asian descent. These disease commonly cause severe pain in sufferers in part due to ischemia caused by the damaged red blood cells blocking free flow through the circulatory system.
SCD is considered a prethrombotic state, since certain characteristics of sickle cells such as abnormal adhesivity and absence of membrane phospholipid asymmetry are involved in the thrombotic process [Marfaing-Koka, et al., Nouv Rev Fr Hamatol 35:425-430, 1993]. Most of the morbidity of SCD appears to be related to the appearance of occlusion of the microvasculature, resulting in widespread ischemia and irreversible organ damage. In addition, pulmonary microthromboemboli have been described in 44 percent of autopsies from thalassemia patients [Chuansumrit et al., J Med Assoc Thai, 76(2):80-84, 1993].
Investigators employing sensitive assays for coagulation have documented signs of a hypercoagulable state resulting in vaso-occlusion in adults with SCD. Vaso-occlusion is a complex process involving cellular, vascular, and humoral factors and possibly thrombotic events. The occurrence of stroke is probably the most devastating complication of SCD in a child [Peters, et al., Thrombosis and Haemostasis 71(2): 69-172, 1994; Tam D., Journal of Child Neurology 12(1):19-21, 1997].
Deficiencies of protein C and enhanced thrombin generation have been reported in patients with SCD or thalassemia [Karayalcin, et al., The American Journal of Pediatric Hematology/Oncology 11(3):320-323, 1989; Hazmi, et al., Acta Haematol 90:114-119, 1993; Peters, 1994]; Shirahata et al. Southeast Asian J Trop Med Pub Health 23(2):65-73] The lower protein C levels in SCD or thalassemia are either due to decreased production or increased consumption. Therefore, a coagulation imbalance exists in patients with SCD or thalassemia which in turn may be responsible for the adverse clinical effects observed in these patients.
Currently, there is no effective therapy to prevent the pain associated with SCD or thalassemia or to correct the disease causing genes. The current treatment approach includes intravenous solutions of glucose and electrolytes, narcotic analgesics, and antiinflammatory agents [Green et al. American journal of Hematology 23:317-321, 1986]. Recently, the chemotherapeutic agent hydroxyurea has been used in an increasing number of sickle cell anemia patients. In more severe cases or following ischemic stroke, exchange transfusions and bone marrow transplantation have been utilized [Ferrera et al. American Journal of Emergency Medicine 15(7):671-679, 1997]. Prophylactic transfusion is the only accepted therapy for patients with SCD that have had a stroke. Therefore, a need exists for a safe, effective therapy of patients with SCD or thalassemia.
The present invention is the first to describe the treatment of SCD or thalassemia with protein C. Protein C, with its anticoagulant, antiinflammatory, and profibrinolytic activities, is useful for the treatment of the hypercoagulable state or protein C deficiency that occurs in SCD or thalassemic patients.
The present invention provides a method of treating a patient suffering from sickle cell disease (SCD) or thalassemia which comprises, administering to said patient a pharmaceutically effective amount of protein C.
The present invention further provides a method of treating sickle cell disease or thalassemia in a patient in need thereof, which comprises administering to said patient a pharmaceutically effective amount of activated protein C such that an activated protein C plasma level of about 2 ng/ml to about 300 ng/ml is achieved.
For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below.
Protein C refers to a vitamin K dependent serine protease with anticoagulant, anti-inflammatory, and profibrinolytic properties which includes, but is not limited to, plasma derived and recombinant produced protein C. Protein C includes and is preferably human protein C although protein C may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant, pro-fibrinolytic, and anti-inflammatory) activities. Examples of protein C derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings of which are hereby included by reference.
Zymogenxe2x80x94an enzymatically inactive precursor of a proteolytic enzyme. Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chains, of protein C.
Activated protein C or aPC refers to protein C zymogen which has been converted by limited proteolysis to its activated form. aPC includes and is preferably human protein C although aPC may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Examples of protein C derivatives are noted above in the description of protein C.
HPCxe2x80x94human protein C zymogen.
r-hPCxe2x80x94recombinant human protein C zymogen.
r-aPCxe2x80x94recombinant human activated protein C produced by activating r-hPC in vitro or by direct secretion of the activated form of protein C from procaryotic cells, eukaryotic cells, and transgenic animals or plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques well known to the skilled artisan and demonstrated in Yan, U.S. Pat. No. 4,981,952, and Cottingham, W097/20043, the entire teachings of which are herein incorporated by reference.
Plasma derived activated protein Cxe2x80x94activated protein C produced by activating plasma HPC as described in Eibl, U.S. Pat. No. 5,478,558, the entire teaching of which is herein incorporated by reference.
Continuous infusionxe2x80x94continuing substantially uninterrupted the introduction of a solution into a vein for a specified period of time.
Bolus injectionxe2x80x94the injection of a drug in a defined quantity (called a bolus) over a period of time up to about 120 minutes.
Suitable for administrationxe2x80x94a lyophilized formulation or solution that is appropriate to be given as a therapeutic agent.
Unit dosage formxe2x80x94refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
Pharmaceutically effective amountxe2x80x94represents an amount of a compound of the invention that is capable of inhibiting sepsis in humans. The particular dose of the compound administered according to this invention will, of course, be determined by the attending physician evaluating the particular circumstances surrounding the case.
The present invention provides for the treatment of sickle cell disease (SCD) or thalassemia with protein C. Protein C, with its anticoagulant, antiinflammatory, and profibrinolytic activities, is useful for the treatment of the hypercoagulable state or protein C deficiency that occurs in SCD or thalassemic patients.
The protein C administered according to this invention may be generated and/or isolated by any means known in the art or as described in U.S. Pat. Nos. 4,981,952, and 5,550,036, herein incorporated by reference. For example, protein C can be produced by secreting full-length, soluble protein C, or biologically active polypeptide variants of protein C from a cell which comprises (a) constructing a vector comprising DNA encoding protein C; (b) transfecting the cell with the vector; and (c) culturing the cell so transfected in culture medium under conditions such that full length soluble protein C or biologically active polypeptide variants of protein C, is secreted. Further, the cell is a eukaryotic cell, e.g. mammalian cell such as Syrian hamster AV12 cell, human embryonic 293 cell, or Baby Hamster Kidney cell.
The protein C used in the treatment of SCD or thalassemia can be formulated according to known methods to prepare pharmaceutically useful compositions. For example, a desired formulation would be one that is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and protein C or aPC.
The protein C will be administered parenterally to ensure its delivery into the bloodstream in an effective form by injecting the appropriate dose as continuous infusion for about 1 hour to about 240 hours.
Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions comprising protein C, as determined by good medical practice and the clinical condition of the individual patient. Generally, the amount of protein C administered will be from about 5.0 xcexcg/kg/hr to about 250 xcexcg/kg/hr. Preferably, the protein C used in the treatment of SCD is activated protein C (aPC). The amount of aPC administered will be from about 1.0 xcexcg/kg/hr to about 96 xcexcg/kg/hr. More preferably the amount of aPC administered will be about 1.0 xcexcg/kg/hr to about 50 xcexcg/kg/hr. While more preferably the amount of aPC administered will be about 1.0 xcexcg/kg/hr to about 35 xcexcg/kg/hr. Even more preferably the amount of aPC administered will be about 5.0 xcexcg/kg/hr to about 30 xcexcg/kg/hr. Yet even more preferably the amount of aPC administered will be about 15 xcexcg/kg/hr to 30 xcexcg/kg/hr. Still even more preferably the amount of aPC administered will be about 20 xcexcg/kg/hr to 30 xcexcg/kg/hr. The preferable amount of aPC administered will be about 24 xcexcg/kg/hr. The most preferable amount of aPC administered will be about 48 xcexcg/kg/hr. The appropriate dose of aPC administered will result in a reduction of the thrombotic complications associated with SCD.
The plasma ranges obtained from the amount of aPC administered will be about 2 ng/ml to about 300 ng/ml. The preferred plasma ranges are from about 2 ng/ml to 200 ng/ml. Most preferably, plasma ranges are from about 30 ng/ml to about 150 ng/ml and still more preferably about 100 ng/ml.
Alternatively, the aPC will be administered by injecting one third of the appropriate dose per hour as a bolus injection followed by the remaining two thirds of the hourly dose as continuous infusion for one hour followed by continuous infusion of the appropriate dose for twenty-three hours which results in the appropriate dose administered over 24 hours. In addition, the bolus injection will be administered via an intravenous bag drip pump or syringe pump at 2 times the normal rate for 15 minutes followed by 1.5 times the normal rate for 45 minutes. The normal rate i.e. that rate which has been determined to administer the appropriate dose level of the therapeutic agent per time period, is then continued for up to 240 hours.
The use of protein C in the treatment of SCD or thalassemia as presented in the present invention will provides a needed therapy for a potentially serious and debilitating disorder. The use of protein C is efficacious and avoids complications such as bleeding tendency, toxicity, and other general side effects of currently available anti-coagulant agents.